During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.

Synapse formation requires a complex interchange of information between the pre- and postsynaptic partners. At the skeletal neuromuscular junction, some of this information is contained in the basal lamina (BL), which runs through the synaptic cleft between the motor nerve terminal and the muscle fibre. During regeneration following injury, components of synaptic BL can trigger several features of postsynaptic differentiation in the absence of the nerve terminal, and of presynaptic differentiation in the absence of the muscle fibre. One nerve-derived component of synaptic BL, agrin, is known to affect postsynaptic differentiation, but no muscle-derived components have yet been shown to influence motor nerve terminals. A candidate for such a role is s-laminin (also called laminin beta 2), a homologue of the B1 (beta 1) chain of the widely distributed BL glycoprotein, laminin. s-Laminin is synthesized by muscle cells and concentrated in synaptic BL. In vitro, recombinant s-laminin fragments are selectively adhesive for motor neuron-like cells, inhibit neurite outgrowth promoted by other matrix molecules, and act as a 'stop signal' for growing neurites. By generating and characterizing mice with a targeted mutation of the s-laminin gene, we show here that s-laminin regulates formation of motor nerve terminals.

Of numerous synaptic components that have been identified, perhaps the best-studied are the nicotinic acetylcholine receptors (AChRs) of the vertebrate neuromuscular junction. AChRs are diffusely distributed on embryonic myotubes, but become highly concentrated (approximately 10,000 microns-2) in the postsynaptic membrane as development proceeds. At least two distinct processes contribute to this accumulation. One is local synthesis: subsynaptic muscle nuclei transcribe AChR subunit genes at higher rates than extra-synaptic nuclei, so AChR messenger RNA is concentrated near synaptic sites. Second, once AChRs have been inserted in the membrane, they form high-density clusters by tethering to a subsynaptic cytoskeletal complex. A key component of this complex is rapsyn, a peripheral membrane protein of relative molecular mass 43K (refs 4, 5), which is precisely colocalized with AChRs at synaptic sites from the earliest stages of neuromuscular synaptogenesis. In heterologous systems, expression of recombinant rapsyn leads to clustering of diffusely distributed AChRs, suggesting that rapsyn may control formation of clusters. To assess the role of rapsyn in vivo, we generated and characterized mutant mice with a targeted disruption of the Rapsyn gene. We report that rapsyn is essential for the formation of AChR clusters, but that synapse-specific transcription of AChR subunit genes can proceed in its absence.

A striking example of topographic specificity in synapse formation is the preferential reinnervation of original synaptic sites on denervated muscle fibres by regenerating motor axons. This specificity is mediated by the basal lamina of the synaptic cleft. A glycoprotein, s-laminin, has now been identified that is selectively associated with synaptic basal lamina and is recognized by motoneurons. Molecular cloning reveals that s-laminin is a novel homologue of laminin, a potent promoter of neurite outgrowth.

S-laminin/laminin beta 2, a homologue of the widely distributed laminin B1/beta 1 chain, is a major component of adult renal glomerular basement membrane (GBM). Immature GBM bears beta 1, which is replaced by beta 2 as development proceeds. In mutant mice that lack beta 2, the GBM remains rich in beta 1, suggesting that a feedback mechanism normally regulates GBM maturation. The beta 2-deficient GBM is structurally intact and contains normal complements of several collagenous and noncollagenous glycoproteins. However, mutant mice develop massive proteinuria due to failure of the glomerular filtration barrier. These results support the idea that laminin beta chains are functionally distinct although they assemble to form similar structures. Laminin beta 2-deficient mice may provide a model for human congenital or idiopathic nephrotic syndromes.